PROJECT SUMMARY This proposal seeks to map and study the function of a likely widespread and potentially powerful modulatory system in the brain that is currently `under the radar' of both basic and clinical neuroscience: the neurosteroid estrogen system. Although estrogens have been studied for decades as reproductive hormones that act through nuclear receptors to regulate gene expression, compelling evidence has accumulated for an additional, distinct system of estrogen action in the brain. In this system, estrogens are produced directly within the brain, of both sexes, where they act through extranuclear receptors to regulate neurophysiology and behavior on a time scale of minutes. Although relatively little is known about the neurosteroid estrogen system, especially in vivo, what is known indicates tremendous potential for neurosteroid estrogens to influence brain functions broadly, from cognition to neuropsychiatric disorders to seizures in epilepsy. Despite this potential, however, current research aimed at understanding where and how neurosteroid estrogens operate in the brain is severely limited by a lack of reliable research tools. We propose to address this problem using a two-pronged approach. First, in a collaborative project, we will generate multiple lines of gene-targeted mice to visualize and map the distribution of key components of the neurosteroid estrogen system: P450 aromatase (estrogen synthase) and three estrogen receptors (ERs), ER?, ER?, and G protein-coupled ER-1 (GPER1), in both males and females. Second, we will adapt approaches for in vivo microdialysis to directly measure steroid levels in specific regions of the male and female brain to identify sites and circumstances of neurosteroid estrogen synthesis. Microdialysis will be followed by targeted manipulation of aromatase activity to investigate the functional consequences of neurosteroid estrogen synthesis. Together, these studies will address five key questions about the neurosteroid estrogen system: (1) Where in the brain are neurosteroid estrogens synthesized? (2) Under what circumstances are they synthesized in vivo? (3) Through what receptors, located where, do neurosteroid estrogens signal? (4) What are the downstream physiological and behavioral consequences of neurosteroid estrogen signaling? (5) How is the neurosteroid estrogen system the same or different in males and females? At the completion of this project, we expect to have generated the first comprehensive maps of the neurosteroid estrogen system in both the male and female brain and to have identified specific behaviors/functions that are influenced by neurosteroid estrogens. Moreover, we expect that this work will transform the field by providing powerful new tools to overcome limitations of current approaches and permit reliable investigation of the neurosteroid estrogen system for the first time.